IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. J. Cell. Sci. (0021-9533) 2004 imex curation Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de orchard Only protein-protein interactions in vitro In vitro electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de Accepted 2009-SEP-3 by ORCHARD. J. Cell. Sci. (0021-9533) 2004 orchard Only protein-protein interactions imex curation Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. J. Cell. Sci. (0021-9533) 2004 imex curation Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de orchard Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de Accepted 2009-SEP-3 by ORCHARD. J. Cell. Sci. (0021-9533) 2004 orchard Only protein-protein interactions imex curation Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. J. Cell. Sci. (0021-9533) 2004 imex curation Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de Accepted 2009-SEP-3 by ORCHARD. J. Cell. Sci. (0021-9533) 2004 orchard Only protein-protein interactions imex curation Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin. J. Cell. Sci. (0021-9533) 2004 imex curation Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de orchard Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Barth M., Holstein SE. susanne_h.holstein@urz.uni-heidelberg.de Accepted 2009-SEP-3 by ORCHARD. J. Cell. Sci. (0021-9533) 2004 orchard Only protein-protein interactions imex curation c0mhr2_pig Clathrin heavy chain CLTC protein protein pig Sus scrofa (Pig) Pig MAQILPIRFQEHLQLQNLGINPANIGFSTLTMESDKFICIREKVGEQAQVVIIDMNDPSNPIRRPISADSAIMNPASKVIALKAGKTLQIFNIEMKSKMKAHTMTDDVTFWKWISLNTVALVTDNAVYHWSMEGESQPVKMFDRHSSLAGCQIINYRTDAKQKWLLLTGISAQQNRVVGAMQLYSVDRKVSQPIEGHAASFAQFKMEGNAEESTLFCFAVRGQAGGKLHIIEVGTPPTGNQPFPKKAVDVFFPPEAQNDFPVAMQISEKHDVVFLITKYGYIHLYDLETGTCIYMNRISGETIFVTAPHEATAGIIGVNRKGQVLSVCVEEENIIPYITNVLQNPDLALRMAVRNNLAGAEELFARKFNALFAQGNYSEAAKVAANAPKGILRTPDTIRRFQSVPAQPGQTSPLLQYFGILLDQGQLNKYESLELCRPVLQQGRKQLLEKWLKEDKLECSEELGDLVKSVDPTLALSVYLRANVPNKVIQCFAETGQVQKIVLYAKKVGYTPDWIFLLRNVMRISPDQGQQFAQMLVQDEEPLADITQIVDVFMEYNLIQQCTAFLLDALKNNRPSEGPLQTRLLEMNLMHAPQVADAILGNQMFTHYDRAHIAQLCEKAGLLQRALEHFTDLYDIKRAVVHTHLLNPEWLVNYFGSLSVEDSLECLRAMLSANIRQNLQICVQVASKYHEQLSTQSLIELFESFKSFEGLFYFLGSIVNFSQDPDVHFKYIQAACKTGQIKEVERICRESNCYDPERVKNFLKEAKLTDQLPLIIVCDRFDFVHDLVLYLYRNNLQKYIEIYVQKVNPSRLPVVIGGLLDVDCSEDVIKNLILVVRGQFSTDELVAEVEKRNRLKLLLPWLEARIHEGCEEPATHNALAKIYIDSNNNPERFLRENPYYDSRVVGKYCEKRDPHLACVAYERGQCDLELINVCNENSLFKSLSRYLVRRKDPELWGSVLLESNPYRRPLIDQVVQTALSETQDPEEVSVTVKAFMTADLPNELIELLEKIVLDNSVFSEHRNLQNLLILTAIKADRTRVMEYINRLDNYDAPDIANIAISNELFEEAFAIFRKFDVNTSAVQVLIEHIGNLDRAYEFAERCNEPAVWSQLAKAQLQKGMVKEAIDSYIKADDPSSYMEVVQAANTSGNWEELVKYLQMARKKARESYVETELIFALAKTNRLAELEEFINGPNNAHIQQVGDRCYDEKMYDAAKLLYNNVSNFGRLASTLVHLGEYQAAVDGARKANSTRTWKEVCFACVDGKEFRLAQMCGLHIVVHADELEELINYYQDRGYFEELITMLEAALGLERAHMGMFTELAILYSKFKPQKMREHLELFWSRVNIPKVLRAAEQAHLWAELVFLYDKYEEYDNAIITMMNHPTDAWKEGQFKDIITKVANVELYYRAIQFYLEFKPLLLNDLLMVLSPRLDHTRAVNYFSKVKQLPLVKPYLRSVQNHNNKSVNESLNNLFITEEDYQALRTSIDAYDNFDNISLAQRLEKHELIEFRRIAAYLFKGNNRWKQSVELCKKDSLYKDAMQYASESKDTELAEELLQWFLQEEKRECFGACLFTCYDLLRPDVVLETAWRHNIMDFAMPYFIQVMKEYLTKVDKLDASESLRKEEEQATETQPIVYGQPQLMLTAGPSVAVPPQAPFGYGYTAPPYGQPQPGFGYSM 6C4F2D54950079E2 ap180_arath Clathrin coat assembly protein AP180 AP180 Clathrin coat-associated protein AP180 T7A14.11 At1g05020 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MPSKLKKAIGAVKDQTSISLAKVANGATGGGDLTTLEVAILKATSHDEEVPIDDRLVTEILGIISSKKSHAASCAAAIGRRIGRTRNWIVALKSLVLVLRIFQDGDPYFPREVLHAMKRGAKILNLSSFRDDSNSCPWDFTAFVRTFALYLDERLDCFLTGKLQRRYTNREQTGRISTNSTTRSRFNPKAGIKSHEPAVRDMKPVMLLDKITYWQKLLDRAIATRPTGDAKANRLVKMSLYAVMQESFDLYRDISDGLALLLDSFFHLQYQSCINAFQACVRASKQFEELNAFYDLSKSIGIGRTSEYPSIQKISLELLETLQEFLKDQSSFPASSGLYPSPNSFLPPPPSSKDSAVSSSLDFGDSTIDTSERYSDYGSFRSTSLEDLMSRTEAGTSSPPMSCHSEPYGGGRDDPNGNNFDTVSTKSLPNNPSVSASNLILDLLSLDDVSNTAEAEDVEDKKKQDDSKAETFDPWEALMLRDDPKKKIETIEEEPSTAEDHQRDSGNWLLALEETATQVQGNNSMAIVPFGLDDPMPAFQAATDQYNPFLEEPVAQLATAGEPMITFGGLALTGFQPEPTFQVNVPDDFEPSSTPTFKATETLPMKCDPFTTFESFGFGETFSENGGVNQQSVLQEQQIWLQNQKKIIAKHLS 6D6B9C9DFE47850A ap2a2_arath AP-2 complex subunit alpha-2 ALPHAC-AD Adaptor protein complex AP-2 subunit alpha-2 Alpha-adaptin 2 Clathrin assembly protein complex 2 alpha-C large chain K5A21.2 At5g22780 Adaptor-related protein complex 2 subunit alpha-2 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MTGMRGLSVFISDVRNCQNKEAERLRVDKELGNIRTCFKNEKVLTPYKKKKYVWKMLYIHMLGYDVDFGHMEAVSLISAPKYPEKQVGYIVTSCLLNENHDFLKLAINTVRNDIIGRNETFQCLALTLVGNIGGRDFAESLAPDVQKLLISSSCRPLVRKKAALCLLRLFRKNPDAVNVDGWADRMAQLLDERDLGVLTSSTSLLVALVSNNHEAYSSCLPKCVKILERLARNQDVPQEYTYYGIPSPWLQVKAMRALQYFPTIEDPSTRKALFEVLQRILMGTDVVKNVNKNNASHAVLFEALSLVMHLDAEKEMMSQCVALLGKFISVREPNIRYLGLENMTRMLMVTDVQDIIKKHQSQIITSLKDPDISIRRRALDLLYGMCDVSNAKDIVEELLQYLSTAEFSMREELSLKAAILAEKFAPDLSWYVDVILQLIDKAGDFVSDDIWFRVVQFVTNNEDLQPYAASKAREYMDKIAIHETMVKVSAYILGEYGHLLARQPGCSASELFSILHEKLPTVSTPTIPILLSTYAKLLMHAQPPDPELQKKVWAVFKKYESCIDVEIQQRAVEYFELSKKGPAFMDVLAEMPKFPERQSSLIKKAENVEDTADQSAIKLRAQQQPSNAIVLADPQPVNGAPPPLKVPILSGSTDPESVARSLSHPNGTLSNIDPQTPSPDLLSDLLGPLAIEAPPGAVSYEQHGPVGAEGVPDEIDGSAIVPVEEQTNTVELIGNIAERFHALCLKDSGVLYEDPHIQIGIKAEWRGHHGRLVLFMGNKNTSPLTSVQALILPPAHLRLDLSPVPDTIPPRAQVQSPLEVMNIRPSRDVAVLDFSYKFGTNVVSAKLRIPATLNKFLQPLQLTSEEFFPQWRAISGPPLKLQEVVRGVRPLALPEMANLFNSFHVTICPGLDPNPNNLVASTTFYSETTGAMLCLARIETDPADRTQLRLTVGSGDPTLTFELKEFIKEQLITIPMGSRALVPAAGPAPSPAVQPPSPAALADDPGAMLAGLL 91A317D5EDD77C25 ap180-at5g22780-1 3 6 unspecified role unspecified role prey prey c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined asp-pro-phe asp_pro_phe608-610ala_ala_ala required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 15 12 direct interaction direct interaction Figure 4 To gain an insight into the existence of a clathrin machinery in plants at the molecular level, the interaction of the two key protein homologs, At-AP180 and At-C-Ad, was investigated. Therefore, H6-At-AP180ENTH was incubated with the two GST constructs of the plant C-adaptin ear region (Fig. 4A). Binding was exclusively observed in the presence of the complete ear region but not with the truncated version of At-C-Ad nor when GST alone was added, showing that the binding site for plant AP180 is located within the extreme C-terminal ear region (760G-1013L) of plant C-adaptin. ap180-at5g22780-2 4 7 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 5 Lowering the amounts of the At-AP180 construct by a factor of four and eight, respectively, reduced binding to the full ear construct, indicating that it is concentration dependent (Fig. 5B). Taken together, the C-terminal region of At-AP180 binds via its 608DPF-motif to the C-terminal ear region of plant C-adaptin. ap180-chc-1 2 5 unspecified role unspecified role neutral component neutral component pig-brain Sus scrofa pig brain brain Brain [cerebrum] 6 unspecified role unspecified role neutral component neutral component c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined undefined mutation asp_pro_phe608-610ala_ala_ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain Wed Jul 06 16:34:26 BST 2016 Sequence change details about this feature cannot be ascertained or do not fit with the current version of the referenced protein, so they have been deleted as a result of our quality control procedures. The original label was 'asp-pro-phe' ecoli Escherichia coli (strain K12) physical association physical association Figure 7 To show their assembly activity, two plant AP180 constructs were used: H6-At-AP180ENTH, which was added in various stoichiometric amounts, and its DLL/AAA-mutated version (GST-At-AP180ENTH-DLL/AAA), which still contains clathrin binding activity. chc-2 2 5 unspecified role unspecified role neutral component neutral component pig-brain Sus scrofa pig brain brain Brain [cerebrum] 5 unspecified role unspecified role neutral component neutral component pig-brain Sus scrofa pig brain brain Brain [cerebrum] physical association physical association Figure 7 Clathrin alone served as a control for spontaneous self-assembly under these conditions. ap180-chc-3 1 5 unspecified role unspecified role neutral component neutral component pig-brain Sus scrofa pig brain brain Brain [cerebrum] 6 unspecified role unspecified role neutral component neutral component c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined undefined mutation asp_pro_phe608-610ala_ala_ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain Wed Jul 06 16:33:52 BST 2016 Sequence change details about this feature cannot be ascertained or do not fit with the current version of the referenced protein, so they have been deleted as a result of our quality control procedures. The original label was 'asp-pro-phe' ecoli Escherichia coli (strain K12) physical association physical association Figure 8 H6-At-AP180ENTH assembles clathrin triskelia into cages of regular size chc-1 1 5 unspecified role unspecified role neutral component neutral component pig-brain Sus scrofa pig brain brain Brain [cerebrum] physical association physical association Figure 8